Ronghua Zhao

Enterochromaffin and serotonin cells are abnormal for patients with colonic inertia

PURPOSE: In recent studies, serotonin and several gut peptides have been shown to serve as regulators of colonic transit. Thus, the distribution, density, and intensity of cells secreting serotonin or certain gut peptides could be abnormal in patients with colonic inertia. The aim of this study was to evaluate the distribution, density, and staining intensity of enterochromaffin and serotonin cells in the colonic mucosa of patients with colonic inertia compared with a control group. METHODS: Between 1993 and 1998 tissue blocks from the right and left side of the colon were obtained in 19 consecutive patients (18 females; mean age, 43.7±11.5 years) who underwent subtotal colectomy for colonic inertia. The control group consisted of colonoscopic biopsies from the right and left colon of 15 patients (all females; mean age, 52.7±16.5 years) for indications other then constipation, inflammatory bowel diseases, or carcinoma. Immunocytochemical staining of enterochromaffin and serotonin cells were performed on 4 µm tissue sections with the primary rabbit antibody against chromogranin A or serotonin, and the biotinylated secondary antibody and enzyme-labeled-streptavidin. The average cell number per microscopic field (×200) was calculated and the proportion of cells with various staining distribution was expressed as the percentage of the entire positive cell population as low, moderate, and high intensity. Studentst-test and chi-squared test were used for statistical analysis, with significance level set atP<0.05. RESULTS: The quantity of both enterochromaffin cells (16.8±10.2) and serotonin cells (12.1±6.4) in the mucosa of the left colon in patients with colonic inertia was significantly higher when compared with the right side of the colon (enterochromaffin cells, 9.4±6.0; serotonin cells, 7.8±3.6;P<0.01). The percentage of both types of cells with low staining intensity was increased, whereas the cells with high and moderate staining intensity were decreased (P<0.01) in the left colon as compared with the right. The number of enterochromaffin cells in left-sided colonic mucosa was significantly higher in the colonic inertia group than in the control group (16.8±10.1vs. 10.4±6.0;P<0.05). Moreover, the numbers of serotonin cells in both the right and left colon was also significantly higher in the colonic inertia group than in the control group (right, 7.8±3.6vs. 4.1±2.4; left, 12.1±6.4vs. 5.8±3.7;P<0.01). In both sides of the colon, the percentage of enterochromaffin and serotonin cells with low staining was significantly higher, whereas percentage of those cells with high or moderate staining was significantly lower in the colonic inertia group than in the control group. In the colonic inertia group there was a significantly positive correlation between numbers of enterochromaffin and serotonin cells (right side,P<0.01; left side,P<0.05). CONCLUSION: In patients with colonic inertia, the number of both enterochromaffin and serotonin cells are significantly increased in the colonic mucosa, especially in the left colon. As indicated by staining distribution, enterochromaffin and serotonin cells contain significantly less hormone than do the same cells in the control group.

Altered serotonin immunoreactivities in the left colon of patients with colonic inertia

Serotonin is an important positive regulator of colonic motility and transit. Its quantity and distribution in the left colon could be abnormal in patients with colonic inertia (CI) and contribute to the disease.

Temporal stability and age-related prevalence of loss of imprinting of the insulin-like growth factor-2 gene

Background: Loss of genomic imprinting (LOI) of the insulin-like growth factor-2 gene (IGF2) is an epigenetic change involving abnormal activation of the normally silent maternally inherited allele. LOI of IGF-2 gene is found in tumor tissue, normal adjoining mucosa and peripheral blood lymphocytes (PBL) of some patients with colorectal cancer (CRC), suggesting that this alteration precedes and is a risk factor for CRC. However, whether LOI of IGF2 is transitory or remains a permanent epigenetic alteration is unknown. Methods: Standard RT-PCR assays for imprinting analysis of IGF2 were performed on PBL from ApaI informative individuals recruited at baseline and repeated 1 to 3 years later. Prevalence of LOI of IGF2 was also evaluated according to age strata. Results: Four-hundred patients, mean age 60.7 years (range 15-95), 287 (80%) Caucasian were studied. This included 210 (51.4%) patients with no colorectal neoplasia, and 190 (48.6) with colorectal neoplasia. LOI of IGF2 was present in all age strata examined, and no statistically significant association across age strata (p trend > 0.05) was noted. Forty-nine patients had repeat analysis of blood imprinting status at a mean follow up time of 38.2 ± 12.9 months. All but three patients had the same imprinting status at follow up (94% agreement, kappa 0.79, p < 0.001). Genomic imprinting was stable for patients with and without CRN. Conclusion: LOI of the IGF2 gene in PBL appears to be a stable epigenetic phenomenon in most patients. Furthermore, LOI of IGF2 was not associated with age, suggesting an inherited or congenital epigenetic event. These findings support the concept that LOI of IGF2 may be a useful risk factor for CRC predisposition.

The differences between American and Chinese patients with Crohn's disease.

Aim  The effect of race on Crohn’s disease (CD) remains uncertain. This study compared the characteristics of American white patients and Chinese patients with CD.

Variability in serotonin and enterochromaffin cells in patients with colonic inertia and idiopathic diarrhoea as compared to normal controls

Aim To evaluate differences in distribution, density and staining intensity of enterochromaffin cells (EC) and serotonin cells (SC) in the colonic mucosa of patients with colonic inertia (CI), idiopathic diarrhoea (ID) and a control group.

Overlapping sphincteroplasty: does preservation of the scar influence immediate outcome?

Background  The importance of the overlapping scar in an anterior sphincteroplasty is often emphasized. The aim of this study was to identify the tissue type used in overlapping sphincter repair based upon ultrasound images, and to correlate these results with the immediate clinical outcome.

Abstract 5362: Analysis of loss of IGF2 genomic imprinting and colorectal cancer risk in Puertorrican Hispanics.

Background: Loss of genomic imprinting (LOI) of IGF-2 has been proposed as an independent risk factor for colorectal cancer (CRC) for Non-Hispanic Whites (NHW) and Asians. However, whether the same association between LOI of IGF2 and CRC risk exist in Hispanics is unknown. Aim: This study aimed at determining the association of LOI of IGF2 in peripheral blood lymphocytes and CRC risk in Hispanics. As a secondary aim we evaluated the clinicopathological characteristics of LOI-positive CRC patients compared to the LOI-negative CRC individuals. Methods: Prospectively recruited patients were classified as informative (heterozygosity for Apa I DNA polymorphism) using TaqMan® SNP Genotyping Assay (Applied Biosystems) on gDNA extracted from peripheral blood lymphocytes. cDNA was synthesized and the imprinting status of IGF2 gene was determined using the same methodology in informative all patients. Demographic, clinical and pathological characteristics were evaluated including: age at diagnosis, gender, tumor size, TNM stage, and differentiation. Statistical analysis was performed using conditional multiple logistic regression models, Odds Ratios (OR) and 95% CI were calculated using STATA 10.0. Results: A total of 587 patients were recruited and analyzed. The cohort was comprised of 359 controls (61.26%) and 227 CRC patients (38.74%) with a mean age 62.1 (range 21-85); and 385 (65.5%) female). Apa 1 analysis identified 222 informative subjects of which 66 individuals had LOI of IGF2 in their PBLs. LOI of IGF2 was not statistically associated with colorectal neoplasia (OR=1.07, 95% CI:0.57-1.99). Furthermore, gender, age at diagnosis of CRC, tumor location, development of prostate, breast or other cancers, and family history of other cancers is not associated with LOI of IGF2. Multiple logistic regression model analysis for the association of CRC and LOI showed that family history of CRC was independently associated with having a personal history of CRC (OR=1.82,95% CI:1.02-3.25). Conclusion: LOI of IGF2 was not independently associated with CRC, colonic adenomas or any of the other cancers examined in this Hispanic cohort. Family history of CRC was the only independent risk factor associated with personal history of CRC. Perhaps other genetic/epigenetic, environmental factors than the ones considered in this study may play a role in the pathogenesis on CRC in the Puerto Rican Hispanic population. Further analysis on genomewide methylation panels is underway to examine the role of epigenetics among Hispanics with CRC. Citation Format: Maria Gonzalez-Pons, Mercedes Y. Lacourt, Sharon Fonseca-Williams, Lorena Marcano, Xiomara Castillo, Ronghua Zhao, Raul Bernabe-Dones, Marcia Cruz-Correa. Analysis of loss of IGF2 genomic imprinting and colorectal cancer risk in Puertorrican Hispanics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5362. doi:10.1158/1538-7445.AM2013-5362

Abstract 1077: Bufalin inhibited cell proliferation and expression of insulin-like growth factor 2 and type 1 receptor in human colorectal cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Objective: To investigate the inhibitory effect of bufalin on the proliferation of human colorectal cancer cell, HCT116, and its relationship with expression of insulin-like growth factor 2 (IGF-2) and type 1 receptor (IGF1R). Methods: HCT116 cells were cultured with bufalin and cell proliferation was measured by MTT assay. The inhibitory rate of cell proliferation and the half maximal inhibitory concentration (IC50) were calculated. Cell cycle was determined by use of flow cytometry. mRNA and protein expressions of IGF-2 and IGF1R were analyzed by real time polymerase chain reaction (PCR) and Western Blot, respectively. The distribution of IGF-2 and IGF1R in cells was also observed by immunocytochemical staining. Results: The IC50 of bufalin at 24, 36, 48, 60, and 72 hours were 0.79±0.10, 0.37±0.10, 0.25±0.03, 0.17±0.02, and 0.10±0.03 uM, respectively. We selected 0.3 uM and 48 hours as the concentration of bufalin and culture time in following experiments. When cultured with bufalin, the ratio of cells in G2 phase to cells in M phase was increased in a dose dependent manner, indicating a delay from G2 to M caused by bufalin. For IGF-2, the mRNA and protein expressions were increased when cells were cultured with bufalin; For IGF1R, the mRNA increased but protein decreased. Both IGF-2 and IGF1R were seen in cytoplasm. Conclusion: Bufalin can significantly inhibit the proliferation of HCT116 cells, its underlying mechanism may relate with the up-regulation of IGF-2 expression and down-regulation of IGF1R protein expression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1077. doi:1538-7445.AM2012-1077

Abstract 1098: Interactions between P53 and IGF2 in human esophageal adenocarcinoma tissues and cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL To define the role of the insulin-like growth factor (IGF) axis in esophageal adenocarcinoma (EADC), the objective of this study was to evaluate interactions between IGF2 and P53 in a well characterized series of surgically-resected esophageal tissues and the following cell lines: Het1A (derived from immortalized normal esophageal epithelium), OE33 and JH-EsoAd1 (each derived from a primary EADC). Nucleic acids were extracted from 68 primary EADC (and matched histologically normal) tissues. PCR was performed on gDNA and RT-PCR on RNA followed by ApaI digestion to identify informative (heterozygous) cases and imprinting status, respectively. Expression of IGF2 mRNA was determined by quantitative PCR, and of IGF2 and P53 protein by Western analysis and immunohistochemistry. Molecular findings in tissues were correlated with pathologic and clinical characteristics including patient survival. Functional studies utilizing cell lines evaluated IGF2/P53 interactions. Of 44 informative cases, P53 protein overexpression was significantly higher in normally imprinted tumors (12/30) vs. tumors with loss of IGF2 imprinting (1/14; p<0.05). Multivariable logistic regression confirmed IGF2 imprinting status to be independently associated with P53 expression (OR 0.12, 95% CI 0.01-1.00; p=0.05). Tumors overexpressing both IGF2 and P53 were found to be of advanced stage with poor survival. In patients below age 65 years, IGF2 mRNA expression was significantly higher in tumors with wild type P53, and multivariate analysis showed IGF2 mRNA expression to be independently associated with p53 mutation (OR 0.74, 95% CI 0.58-0.94; p<0.05) and P53 protein expression (OR 0.83, 95% CI 0.69-1.00; p<0.05). Whereas treatment of Het1A cells with IGF2 significantly increased P53 expression (1.34+/-0.06 vs. 1.00+/-0.00 untreated cells; p=0.003), IGF2 did not modulate P53 expression in either of the two tumor cell lines (OE33 and JH-EsoAd1). IGF1 Receptor (IGF1R) inhibition with AG1024 reduced P53 expression only in OE33 (0.48+/-0.22 vs. 1.00+/-0.00 untreated cells; p=0.05) and JH-EsoAd1 (0.71 +/- 0.23 vs. 1.00+/- 0.00 untreated; p = 0.15) cells, an effect not reversed by IGF2 treatment. In conclusion: 1) these studies identify novel molecular regulatory interactions between P53 and IGF2 in esophageal malignancy, which are dependent on IGF2 imprinting status and tissue/cell type. 2) Overexpression of both IGF2 and P53 in esophageal tumors is associated with aggressive disease in younger patients (below age 65 years), and may represent clinically useful biomarkers to define a biologically distinct subset of EADC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2011-1098

Abstract 1092: Insulin-like growth factors modulate chemosensitivity to cisplatin and 5-fluorouracil in human esophageal cell lines

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Resistance to chemotherapy remains a major obstacle to survival of patients with esophageal adenocarcinoma (EADC). The aim of this study was to evaluate whether insulin-like growth factors (IGFs), recently implicated in the molecular pathogenesis of EADC, modulate chemosensitivity to Cisplatin (CDDP) and 5-Fluoruracil (5-Fu), two agents widely used in current clinical practice. Expression of IGF1, its receptor (IGF1R), and IGF2 (mRNA and protein) were studied using quantitative (real-time) PCR and Western blot in human esophageal cell lines: Het1A (derived from immortalized normal esophageal epithelium), OE33 and JHEsoAd1 (each derived from a primary EADC). For each cell line, the effect of various concentrations of IGFs, CDDP and 5-Fu, alone and in combination, on cell proliferation and clonogenicity were evaluated by standard MTT and colony formation assays, respectively. Changes in expression of 84 critical genes downstream from IGF1R were studied by PCR-array. Relative to Het1A, IGF1 and IGF2 mRNA were significantly (P<0.05) underexpressed by OE33; by contrast, JHEsoAd1 underexpressed IGF1 but overexpressed IGF2. Administration of either IGF1 (250ng/ml) or IGF2 (500ng/ml) to cell cultures resulted in increased cellular proliferation of JHEsoAD1 cells (after IGF1: 1.24+/-0.05 vs. 1.00+/-0.00 untreated, P<0.01; after IGF2: 1.15+/-0.06 vs. 1.00+/-0.06 untreated, P<0.05). As expected, cell proliferation and clonogenicity of all cell lines were inhibited by CDDP and/or 5-FU (at all dosages ranging from 1-4ug/ml). However, these cytotoxic effects were overcome by the administration of either IGF1 or IGF2 with the exception of: 1) JHEsoAd1 cells treated with CDDP, where IGF2 administration further reduced cellular proliferation (0.35+/-0.06 vs. 0.53+/-0.03 for cells treated with CDDP alone; P<0.05), and 2) JHEsoAd1 cells treated with 5-Fu, where IGF1 administration significantly reduced clonogenicity (0.07+/-0.07 vs. 0.49+/-0.06 for cells treated with 5-Fu alone; P<0.05). PCR-array analysis revealed up-regulation (Het1A and JHEsoAd1) and down-regulation (OE33 and JHEsoAd1) of selected PI3K/AKT pathway genes following IGF1 and IGF2 therapy. These pre-clinical studies confirm a central role for the IGF axis in esophageal tumor biology, with potential for IGFs to enhance the cytotoxic efficacy of standard chemotherapeutic agents in esophageal cell lines. The identification of novel molecular regulatory pathways downstream from IGF1R modulated by IGF therapy may well inform future cytotoxic and targeting strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1092. doi:10.1158/1538-7445.AM2011-1092